Screening The Proteins Associated With Early Lung Squamous Carcinoma And Validating On Differential Expression

Lung cancer is the leading cause of cancer-related death worldwide and the incidence and mortality shows an obvious upward trend.At present lung cancer is the largest carcinoma in China and becomes the priority for the prevention and treatment of tumor.Early diagnosis and treatment is the effective way to reduce the mortality and prevent for lung cancer.However,early diagnosis is difficult to resolve because of the asymptomatic manifestations,and the foundation to resolve this problem is to searching the gene or protein markers,that is significant signs in the development of lung cancer. The researches on the molecular biology show that the incidence and development of lung cancer is a process of multistage,multi-step,multifactor involved abnormal changes in a large number of gene and proteins.In certain degree lung cancer is considered as a disease with exceptional proteins.Therefore it has theoretical and practical significance for early diagnosis and treatment to look for molecular markers with high specificity and sensitivity from protein level.Proteomics method has been widely used to find protein markers associated with tumor,and the classical method is comparative proteomics which compares tumor tissue/cell/serum in case and control,then screening the differential proteins in different states.However,the credibility of the experimental results could decline because of the mesenchymal interference existing in tumor tissue.Serologic proteome analysis(SERPA) is a new method of proteome technology from the perspective of humoral immune which combines the two-dimensional gel electrophoresis(2-DE) with Western blot.Tumor mesenchymal tissue with no significant specificity could basically not stimulate the body to produce antibodies,so SERPA can avoid the false positive interference caused by mesenchymal tissue to a certain extent.However,screening cancer-related protein by means of antibodies has the shortcoming of instability in sensitivity and specificity because of the limitations of serological examination.Combining comparative proteomics with SERPA can greatly enhance the accuracy and reliability of proteome analysis methods,and it maybe more desirable for screening tumor markers.Most of previous proteomics researches are often on the conclusions of mass spectrometry.The entire process from the 2-DE technology to the mass spectrometry analysis is too long with many influencing factors,although some differential proteins associated with lung cancer have been screened and identified,expressing status and the role still needs to be further studied,and the different comparison also need to validate in various levels.Therefore,in order to ensure the credibility of screening and further scientific research,it is necessary to verify the results of 2-DE.Screening the biomarkers with high specificity and sensitivity is the important way for early diagnosis and treatment of lung cancer.However,the current reports still have shortcomings.On the one hand the existing molecular markers are basically targeted at the advanced stage of tumor,so the incidence of cancer is not really reduced.On the other hand the different pathological types of lung cancer have various pathogenesis,the single pathological type of research will be more accurate.In order to screen and identify proteins associated with early lung squamous cell carcinoma,tissues of lung squamous carcinoma inⅠstage and corresponding adjacent normal tissue,sera of lung cancer patients and normal health were collected as objects and materials.Comparative proteomics combining with SERPA were used to screen out proteins associated with early lung squamous carcinoma.This study would enrich the awareness of human lung tissue proteins,provide the basis for early diagnosis through searching the specific proteins by identifying and validating the differential proteins,and also help to further reveal the molecular mechanism in development of lung cancer.Objective1.Establishing the stable technology platform for comparative proteomics and SERPA by optimizing two-dimensional gel electrophoresis and Western Blot,the proteins associated with early lung squamous carcinoma were analyzed by MALDI-TOF-MS to screen the proteins with potential value.2.The expression of differential proteins were detected by real-time quantitative PCR (RQ-PCR),Western Blot and immunohistochemical method to validate the reliability and accuracy of the results of mass spectrometry.At the same time the clinical and pathological characters were analyzed to explore the change of them in the development of lung cancer.Materials and methods1.SubjectsTissue sample:The respective 22 tissue samples of lung squamous carcinoma and adjacent non-cancerous were collected and confirmed with histopathology.The samples included 6 cases at stageⅠ,4 cases ofⅡstage,and 12 cases ofⅢstage;5 cases of high differentiation,the 7 cases of middle differentiation,and 10 cases of poor differentiation.Sera sample:12 cases of lung squamous cancer patients and healthy respectively.2.Methods2.1 Screening and identifying of proteins associated with lung squamous carcinoma2.1.1 Sample preparationThe soluble proteins of respective 6 tissue samples of lung squamous carcinoma in stage I and adjacent non-cancerous were extracted with lysis solution of 2-DE ingredient. Protein concentrations of the samples were determined by the Bradford assay as the loading amount basis of analytical and preparative gels.2.1.2 Comparative proteomicsFirst the samples in different groups were mixed with equal amount of each one respectively,the protein with analytical sample volume(11cm pH3-10 IPG strip) is separated by 2-DE under the optimized conditions.Each mixture in case and control groups were conducted three times of 2-DE under the same conditions,and the analytical gels were obtained after silver staining.The stained gels were scanned by ImageScanner to obtain the images and analyzed by ImageMaster 2D platinum 6.0 Software.2.1.3 SERPAThe 2-DE gels for the soluble proteins of lung squamous carcinoma tissues including 3 parallel gels were obtained with protein standard quantity for preparative gel(11cm pH3-10 IPG strip),then were transferred to nitrocellulous membranes.After having reacted with 3 groups of mixed sera from lung squamous carcinoma patients and from normal healthy persons,the nitrocellulose(NC) membranes were incubated with goat anti human IgG as second antibody,DAB kit was used to reveal the result of immunoreactivity.Finally the differential Western Blot maps were established.After scanning gels and the Western Blot images with ImageMaster,the data were analyzed with ImageMaster 2D platinum 6.0 Software and manual comparison to acquire the differential protein spots and identified their differential corresponding spots in gel.The differential proteins were located in parallel gel.The common differential proteins between the analytical maps of comparative proteomics and the profiles of differential corresponding proteins were selected as differential proteins.2.1.4 Identification of the differential proteins by MSThe differential spots were cut out from preparative gel(17cm pH3-10,IPG strip) and then digested with typsin.Peptide Mass Fingerprint(PMF) or peptide Sequence Tag (PST) was obtained by matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and tandem mass spectrometry(TMS).The protein database was used to search and identify the protein identities as the candidate proteins of tumor-associated antigens in lung squamous carcinoma tissue.2.2 Validating of ANXⅠ,HSP27 and Ran associated with lung squamous carcinoma2.2.1 Validating on gene level by FQ-PCRThe total RNA was extracted from lung squamous carcinoma and adjacent non-cancerous tissues,and cDNA was synthesized by reverse transcription.ANXⅠ, HSP27 and Ran were amplified by FQ-PCR to analyze the expression and the relationship between the expressing difference and clinical features and tumor pathological information of patients.2.2.2 Validating on protein level by Western BlotThe soluble proteins of respective 22 tissue samples of lung squamous carcinoma and adjacent non-cancerous were extracted,and the concentrations were measured by the Bradford assay.The appropriate sample volume was taken to conduct SDS-polyacrylamide gel electrophoresis,subsequently the separated proteins were transferred to NC membranes.After having reacted with specific primary antibody,the NC membranes were incubated with goat anti human IgG as second antibody.ECL was used to reveal the result of immunoreactivity,finally the expression level of protein was analyzed.2.2.3 ImmunohistochemistryThe tissues of squamous carcinoma and corresponding adjacent normal tissues were fixed by formalin and embedded by paraffin,then cut with continuous sections. Immunohistochemistry staining was carried out by Streptavidin /Peroxidase(SP) to detect the protein location and expression. 3.Statistical analysisData analysis was performed by means of SPSS 12.0 software package with the level of testα=0.05.T test was adopted to analyze the expression levels of gene and protein, and the chi square test was used to analyze the ratio.Results1.Screening and identifying of the differential proteins1.1 The proteomic profile with higher resolution and repeatability of lung squamous cell carcinoma and the adjacent normal tissue was established.After three times of 2-DE for mixture of each group,the protein expression profiles were conducted matching analysis which considered the map with best separated quality and most spots of each group as the reference gel.In the group of lung squamous cell carcinoma the average matched protein spots and average matched rate were 626 and 87.46%respectively.In the group of adjacent normal tissue the average 602 protein spots matched were obtained and the average matched rate was 89.21%.Based on the matching analysis and combined with observation and comparison 38 differential proteins were screened.1.2 The Western Blot maps were obtained through the immunoreactions including lung squamous carcinoma tissues and mixed sera from both lung squamous carcinoma patients and from normal health persons.From the maps 16 differential spots were screening by image software analysis and artificial observation,after that they were located in parallel gel.Combined with the differential proteins in comparative proteomics maps,10 common protein spots were screened.1.3 Ten protein spots were cut out from the preparative gel and were identified with MALDI-TOF-MS.After searching the protein database,the 10 proteins were identified out Successfully:annexinⅠ(ANXⅠ),heat shock protein 27(HSP27),ras-related nuclear protein(Ran),S100 calcium-binding protein A9(S100A9),ferritin light polypeptide(FLP),peroxiredoxin 3(PRX3),Enoyl Coenzyme A hydratase 1(ECH1),elongation factor(EF-Tu),poly cR binding proteinⅠ(PCBP1) and PWP1-interacting protein 4.2.Results of validating of ANXⅠ,HSP27 and Ran2.1 Results of validating of ANXⅠThe results of Fluorescence quantitative RT-PCR showed the mRNA expression of ANXⅠin squamous cell carcinoma was 1.81 times of that in the adjacent normal tissues, the difference was significant(P＜0.05).The level of mRNA expression in poorly differentiated tissues was higher than the middle and high differentiation tissues in lung squamous cell carcinoma.The expressing difference between TNM stages was not found (P＞0.05).Western blot analysis showed that the expression of ANXⅠin squamous cell carcinoma was higher than that in adjacent normal tissues.ANXⅠprotein expressing level in poorly differentiated carcinoma tissues was higher than that in high and middle differentiation cancer tissues,the difference was significant(P＜0.05).Between TNM stages the expressing difference was not found.The expression between mRNA and protein was consistent.The results of immunohistochemistry showed that the ANXⅠmainly located in the membrane and the cytoplasm,and not found the location change between lung squamous cell carcinoma and adjacent normal tissue.The positive expression rate and strong positive rate of ANXⅠin lung squamous cell carcinoma tissues were 72.73%(16/22) and 40.91%,and in adjacent normal tissues were 31.82% (7/22) and 9.09%respectively.Compared with adjacent normal tissue,the positive rate and strong positive rate significant increased in lung squamous cell carcinoma.The change on protein expression level had the same trend with 2-DE and verified the 2-DE results.2.2 Results of validating of HSP27The results of Fluorescence quantitative RT-PCR showed that the difference of HSP27 mRNA expression in squamous cell carcinoma and adjacent normal tissues was not significant(P＞0.05).Western Blot analysis showed that the expression of HSP27 in squamous cell carcinoma was higher than that in adjacent normal tissues.HSP27 protein expressing level in poorly differentiated carcinoma tissues was higher than that in high and middle differentiated cancer tissues,the difference was significant(P＞0.05).Between TNM stages the expression difference was not found(P＞0.05).The results of immunohistochemistry showed that the HSP27 mainly located in the membrane,and not found the location change between lung squamous cell carcinoma and adjacent normal tissue.The positive expression rate and strong positive rate of HSP27 in lung squamous cell carcinoma tissues were 72.27%(17/22) and 31.82%(7/22),and in adjacent normal tissues the positive rate was 18.18%(4/22) and strong positive expression was not found. Compared with adjacent normal tissue,the positive rate and strong positive rate significant increased in lung squamous cell carcinoma(P＜0.05).The change on protein expression level had the same trend with 2-DE and verified the 2-DE results.2.3 Results of validating of RanThe results of Fluorescence quantitative RT-PCR showed that Ran gene mRNA expression levels in squamous cell carcinoma was 2.35 times of that in the adjacent normal tissues,the difference was significant(P＜0.05).The mRNA expressing level ofⅢstage was higher than theⅠandⅡstage tissues in lung squamous cell carcinoma.The difference of expressing level between differentiation degree was not found(P＞0.05). Western Blot analysis showed that the expression of Ran in squamous cell carcinoma was higher than that in adjacent normal tissues.The protein expression level ofⅢstage tissues was higher than theⅠandⅡstage in lung squamous cell carcinoma(P＜0.05).The difference of expression level between differentiation degree was not found(P＞0.05).The results of immunohistochemistry showed that Ran mainly located in the nuclear,and not found the location change between lung squamous cell carcinoma and adjacent normal tissue.The positive expression rate and strong positive rate of Ran in lung squamous cell carcinoma tissues were100%(22/22) and 72.73%(16/22),and in adjacent normal tissues were 59.09%(13/22) and 4.55%respectively.Compared with adjacent normal tissue,the positive rate and strong positive rate significant increased in lung squamous cell carcinoma(P＜0.05).The change on protein expression level had the same trend with 2-DE and verified the 2-DE results.Conclusion1.10 candidate proteins of tumor-associated antigens in lung squamous carcinoma tissue were screened out and identified with MS combining application of comparative proteomics and serologic proteome analysis.2.The protein level of ANXⅠ,HSP27 and Ran expression in lung squamous cell carcinoma was higher than that in adjacent normal tissues respectively.The trends of expression changes of them were consistent with 2-DE.These proteins maybe played an important role in process of development of lung cancer.Ran maybe become an importmant marker for early diagnosis.